Containers for chromatography media

ABSTRACT

The invention relates to containers or bags for chromatographic media and methods of packing chromatography columns using such containers. The bags may be used for storing and/or transporting chromatographic media and can be inserted directly into the chamber of a chromatography column in readiness for use.

FIELD OF THE INVENTION

The invention relates to the separation of chemical compounds by columnchromatography. In particular, the invention relates to containers orbags for chromatographic media and methods of packing chromatographiccolumns or preparing consolidated beds of chromatographic media usingsuch containers.

BACKGROUND

Columns used in liquid chromatography typically comprise a tubular orcylindrical body enclosing a packed bed of porous chromatography mediumthrough which a carrier liquid flows, with separation taking place bypartitioning between the carrier liquid and solid phase of the porousmedium. Cylindrical columns are generally known as ‘axial’ columns,chromatographic separation typically occurring in a vertical directiondown the length of the column, while tubular columns are generally knownas ‘radial’ columns with separation taking place in a radial directionas the carrier liquid flows to the centre of the cylinder.

Prior to any separation process, the bed has to be prepared by startingfrom the particulate medium that is to be introduced into the column.Conventional columns aimed for high efficiency separations deploy aprocess of bed formation, which is called ‘the packing procedure’. Inhigh efficiency separations, a correctly packed bed is a critical factorinfluencing the performance of a column containing a packed bed.Typically, the packed bed is prepared by slurry packing, i.e.

consolidating a suspension of discrete particles or fibres in liquid,known as slurry that is pumped, poured, or sucked into the column. Oncethe predetermined volume of slurry has been delivered into the column itneeds to be further consolidated and compressed.

In axial columns or in axial chromatography, the slurry can becompressed by moving a movable adapter down the longitudinal axis of thecolumn towards the bottom of the column, normally at a constant speed.The excess liquid during this procedure is expelled at the columnoutlet, while the media particles are retained by means of a filtermaterial, a so-called ‘bed support’ or ‘frit’, with pores too small toallow the media particles to pass though. The packing process iscomplete once the packed bed has been compressed by the optimum degreeof compression.

Another approach for column slurry packing used both in axial and radialcolumns is the flow packing method, where compression of the porousstructure is primarily achieved by applying a high flow rate over thecolumn, thereby forming a porous structure starting at the outlet bedsupport. The resulting drag force on the particles in the porousstructure causes eventually a pressure drop and a compression of thebed. The compressed bed is finally confined by bringing the adapter intoposition. Presently, only flow packing methods are known for packingradial columns.

The efficiency of subsequent chromatographic separation relies stronglyon 1) the liquid distribution and collection system at the fluid inletand outlet of the packed bed, 2) the special orientation (also know asthe packing geometry) of the media particles in the packed bed, and 3)the compression of the packed bed. If the compression of the packed bedis too low then chromatographic separations performed on that bed sufferfrom “tailing” and, generally, such insufficiently compressed beds areunstable. If the compression of the packed bed is too high thenchromatographic separations performed by the bed suffer from “leading”and such over-compressed beds can affect throughput and bindingcapacity, and, in general, give much higher operating pressures. If thecompression is optimum, then the separation peaks formed during useexhibit much less leading or tailing and are substantially symmetrical.The optimum degree of compression is also crucial for achieving goodlong-term stability of the porous structure, thereby securing optimalperformance throughout a number of process cycles. The optimum degree ofcompression required for a column is determined experimentally for eachcolumn size (width or diameter), bed height, and media type.

An alternative packing method used for axial columns is called “drypacking”, where the column is filled with dry particles of the porousmedium and liquid is introduced in the column afterwards. This hasadvantages in prepacked columns that can be delivered dry to thecustomer without having to add any preservatives to the packing liquidand minimizing weight during transport. Dry packing is typically usedfor silica media aimed at separation of small molecules, as describede.g. by G Guiochon J Chromatogr A 704 (1995) 247-268, although fairlypoor column efficiencies are obtained. For swellable chromatographymedia, such as dextran or agarose-based media commonly used inseparation of biomolecules, dry packing has however been avoided due toa perception that the swelling of the particles will cause poorperformance of the packed bed.

The above described background for preparation of packed beds adaptedfor high efficiency chromatographic separations applies equally topacked beds or fixed beds aimed for achieving bio-reactions such asenzymatic conversions, the treatment of cells or the growth andcultivation of cells adherent to the packed bed with high efficiency.

The columns and packed beds described above may also be used forstabilisation of substances and in particular biomolecules such asproteins, antibodies and cells. This stabilistation may improve andfacilitate storage and transport of the molecules, for example. Thebinding of substances, particularly biomolecules, to chromatographymedia may preserve activity and stability of the substance more than isachievable with other concentration methods such as filtration,precipitation or freeze-drying.

TECHNICAL PROBLEM

There are a number of problems associated with existing methods forpreparing and packing columns. Media is generally shipped to operatorsin large containers and must be carefully weighed out and transferred tocolumns in preparation for the packing procedure. This can be atime-consuming, wasteful and expensive process as it involves a trainedtechnician as much of the media is not used and must be stored. Wherethe media are to be used in the preparation of approved products, suchas drugs and foodstuffs, regulatory requirements must also be satisfied.Sterilisation and cleaning of the media and/or column to reducemicrobial load can prove difficult for users. Furthermore, the columnsare often complex and have a large ‘footprint’ in order to carry out thepacking procedure.

Another problem is that columns known in the prior art are not suitablefor the stabilisation of substances for the purpose of storage andtransport due to high cost, complexity and low flexibility. There istherefore a need to provide a simple and cost-efficient device andmethod for accomplishing a stabilisation of substances through theprocess of adsorption to a chromatography medium and desorption prior tofurther processing or use.

It is an object of the present invention to mitigate the aforementionedproblems associated with prior art methods of packing columns andstabilising substances for storage and transport.

SUMMARY OF THE INVENTION

In a first aspect of the present invention, there is provided a flexiblebag for chromatographic medium, said bag comprising an exterior wall ofa non-porous material attached to a first liquid distribution elementand a second liquid distribution element thereby defining a compartmentfor chromatographic medium therein; said first liquid distributionelement being opposed to said second liquid distribution element; and amedium filling port; optionally comprising a first and a second endpiece attached to said exterior wall and adjacent to the first andsecond liquid distribution element respectively, both said end piecescomprising an opening for receipt of an inlet or an outlet for carrierliquid; wherein the first liquid distribution element and/or the secondliquid distribution element are welded or moulded to said wall.

In one aspect, the bag has a tubular configuration and additionallycomprises an interior wall, wherein the first liquid distributionelement is welded or moulded to said exterior wall and the second liquiddistribution element is welded or moulded to said interior wall.Preferably, the bag is for use in radial chromatography.

In another aspect, the bag has a cylindrical configuration and comprisesa first and a second end piece attached to the exterior wall andadjacent to the first and second distribution element respectively, bothsaid end pieces comprising an opening for receipt of an inlet or outletfor carrier liquid.

In a further aspect, the first and second end piece are welded ormoulded to the exterior wall. Preferably, the first and second end pieceare made from a rigid or semi-rigid material. Suitable materials includeinert metals or plastics.

In one aspect, the first liquid distribution element and the secondliquid distribution element is selected from the group consisting offilter, mesh and net. Preferably, the distribution element is a woven,mesh, filter and sinter.

In another aspect, the first liquid distribution element and the firstend piece are an integrated unit, and the second liquid distributionelement and the second end piece are an integrated unit. The integratedunit may be formed, for example, by moulding or machining Suitably, thefirst end piece portion is made from polypropylene.

In a further aspect, the bag is for use in axial chromatography.

In one aspect, the bag additionally comprises a second compartmenthaving an inlet for hydraulic fluid.

In another aspect, the wall or film is made from a plastic polymericmaterial, in particular materials which are for welding or moulding.Plastic polymers based upon polyethylene are particularly suitable. Thussuitable walls or films include, for example, American Renolit InfuflexBarrier 9101 (SOLMED®. BF 9101; American Ronolit Crop, La Porte, Ind.,USA), American Renolit BF-1400 (SOLMED® BF-1400 Film; American RenolitCorp, La Porte, Ind., USA), American Renolit 4301 (SOLMED® Granuflex,American Renolit Crop, La Porte, Ind., USA), Mitos Durapure (MitosTechnologies, Phoenixville, Pa., USA) and MILLIPORE® Pureflex ProcessContainer Film, (Millipore Corp, Billerica, Mass., USA).

In a further aspect, the bag additionally comprises a semi-rigid orrigid housing around the wall to provide rigidity in an axial and/orradial direction.

In one aspect, the bag additionally contains wet or dry chromatographicmedium. In a preferred aspect, the wet or dry chromatographic medium issterile. The bag can be sterilised by treatment with radiation,autoclaving or chemical disinfectants. Gamma radiation is particularlyeffective. Milder treatments can also be used to reduce the microbialload, or total number of microbes such as bacteria and fungi present inthe medium.

In a second aspect of the present invention, there is provided a methodof packing a chromatography column, said method comprising the steps of

a. inserting a bag as hereinbefore described into a chamber of achromatography column;

b. closing the column housing; and

c. forming or compressing a packed bed or a consolidated bed or afluidised bed of chromatographic medium.

The consolidated bed is here defined as a porous structure of a bed ofchromatography medium in the absence of a substantial mechanicalcompression. A consolidated bed is for example formed by applying adownward fluid flow to an axial chromatography column filled with asuspension of chromatography medium. Hereby, a porous consolidated bedis formed at the outlet of the column (at the filter) until allchromatography medium has been settled. Such a consolidated bed can beused to achieve a binding of a chemical substance, such as a protein,peptide, antibody, nucleic acid or other biological molecule. Theconsolidated bed can also be used to bind cells.

The top end piece/filter may be fixed (gap formation) or moved towardsor close to the surface of the consolidated bed to reduce the hold-upvolume and gap, respectively.

One aspect of the invention is to provide a flexible container for aconsolidated bed where one or a plurality of flexible walls aremechanically supported by a rigid housing for providing rigidity whenapplying fluid pressure to the interior space of the container and theconsolidated bed.

Another aspect of the invention is to provide a flexible container thatcontains chromatography medium and where flexible container with itscontent can be frozen after having loaded the chromatography medium witha chemical.

Another aspect of the invention is to provide a flexible containerfilled with chromatography medium that can be frozen, thawed and used toform a consolidated bed from the chromatography medium in order to applyfurther process steps involving the application of liquid to saidconsolidated bed.

In one aspect, the bag contains dry chromatographic medium and saidpacked bed is formed by adding liquid to swell said medium to give aswollen volume Vs in a liquid of about 105-120% of the column chamber.The term “swollen volume (Vs)” as used herein means the sediment volumeof an aliquot of particles suspended and equilibrated in a liquid. Thesediment volume can be measured by suspending the aliquot of particles,equilibrating for up to 24 hours, resuspending the particles if needed,letting the particles sediment and measuring the sediment volume in agraded vessel such as a measuring cylinder.

In another aspect, the bag contains wet medium, the column housing isrigid and the packed bed is formed or compressed by axial compression ofthe bag.

In a further aspect, the column comprises a hydraulic chamber and thepacked bed is formed or compressed by filling said chamber with ahydraulic fluid. The hydraulic chamber may be a flexible bag. In oneaspect, the bag may comprise a second compartment and the packed bed isformed or compressed by filling said second compartment with a hydraulicfluid.

Optionally, the column comprises a movable piston or adapter and thepacked bed is formed or compressed by axial movement of said piston oradapter.

In one aspect, the bag containing wet medium is placed over a rigid corewithin the column chamber and the packed bed is formed or compressed byradial compression of the bag. Preferably, the packed bed is formed orcompressed by radial compression of the column housing. In one aspect,radial compression of the column housing is effected by tighteningradial bands on the outside of the housing.

In another aspect, the volumetric compression of the wet medium is 5 to20%. Preferably the volumetric compression is 7.0 to 15%.

In a further aspect, the method additionally comprises sterilising thecolumn with radiation, such as gamma radiation. Similar treatments canbe used to reduce the microbial load.

In one aspect, a consolidated or a fluidised bed of chromatographicmedium is formed, the method additionally comprising the steps of d)loading a chemical or a cell onto said consolidated bed or fluidisedbed; and e) removing said bag from said chamber of said chromatographycolumn for storage and/or transport. In a preferred aspect, the chemicalis selected from the group consisting of protein, antibody, peptide,oligopeptide, nucleic acid, oligonucleotide, RNA, DNA, oligosaccharideand polysaccharide.

In another aspect, the method additionally comprising the step ofstoring and/or transporting the bag. The bag is stored and/ortransported at a temperature between −20.degree. C. and 20.degree. C.

In a further aspect, the method additionally comprising the step ofreturning the bag to the chromatography column and eluting said chemicalor cell from said consolidated bed or fluidised bed. In a preferredaspect, the chemical is selected from the group consisting of protein,antibody, peptide, oligopeptide, nucleic acid, oligonucleotide, RNA,DNA, oligosaccharide and polysaccharide.

In a third aspect of the present invention, there is provided a use of abag according to the first aspect of the invention for storing and/ortransporting a chemical or a cell loaded onto wet or dry chromatographicmedium. In one aspect, the bag is stored and/or transported at atemperature between −20.degree. and 20.degree. C. centrigrade.

In a fourth aspect of the present invention, there is provided achromatograpy column comprising a flexible bag as hereinbeforedescribed.

According to a fifth aspect of the present invention, there is provideda use of a chromatography column as hereinbefore described forseparating or purifying or processing a chemical or a cell. In apreferred aspect, the chemical is selected from the group consisting ofprotein, antibody, peptide, oligopeptide, nucleic acid, oligonucleotide,RNA, DNA, oligosaccharide and polysaccharide. Preferably,

BRIEF DESCRIPTION OF THE DRAWINGS

FIGS. 1a and 1b show schematic, sectional views of one embodiment of awelded bag (10) according to the invention that has a semi-ridgedhousing that can be packed by axial compression.

FIGS. 2a, b and c are schematic sectional plan views of a cartridgecontaining a bag in accordance to the invention which can be packed byradial compression.

FIGS. 3a, b and c are schematic diagrams illustrating the componentparts of a column which contains a bag according to the invention whichcan be packed by radial compression. FIGS. 3d and e illustrate how thecomponent parts are fitted together and operate.

FIGS. 4a, b and c show results from low temperature storage experimentswith pH 7.4, 5.0, and 9.0 respectively.

DETAILED DESCRIPTION OF THE DRAWINGS

FIGS. 1a and 1b show schematic, sectional views of one embodiment of awelded bag (10) according to the invention placed in a column (20). Thebag (10) consists of a first (13) and a second (14) liquid distributionelement welded to a wall (12) of a non-porous material. The distributionelements (13, 14) are bed supports or frits or nets or sinters which actas filters, having pores that are too small to allow chromatographicmedia to pass through but are porous to liquids. The non porous materialor film is made of a plastic polymer, those based upon polyethylenebeing particularly suitable for welding or moulding. For example,suitable films include American Renolit Infuflex Barrier 9101 (SOLMED®BF 9101; American Ronolit Crop, La Porte, Ind., USA), American RenolitBF-1400 (SOLMED® BF-1400 Film; American Renolit Corp, La Porte, Ind.,USA), American Renolit 4301 (SOLMED® Granuflex, American Renolit Crop,La Porte, Ind., USA), Mitos Durapure (Mitos Technologies, Phoenixville,Pa., USA) and MILLIPORE® Pureflex Process Container Film, (MilliporeCorp, Billerica, Mass., USA).

The bag also comprises a chromatography medium filling port (16) throughwhich dry or wet media or slurry is added to the bag (10). In theembodiment shown in

FIG. 1b , the bag (10) has one compartment (11) for receipt of media(not shown) via port (16). Although not shown in FIG. 1a or 1 b, the bag(10) also comprises a first and second end piece attached to theexterior wall (12) and adjacent to each of the distribution elements(13, 14), respectively. The end pieces are made from a rigid orsemi-rigid material that prevents seepage of carrier liquid from the bag(10) and has openings for receipt of an inlet (25) and outlet (26) forcarrier liquid. In one embodiment, the opening takes the form of aresilient septum (not shown) which receives the inlet (25) or outlet(26) for carrier liquid. Suitable materials for the production of theend pieces include inert metals and plastics such as polypropylene.Preferably the end piece is welded or moulded to the exterior wall (12).

In one embodiment (not shown) the end piece and the distribution elementmay be integrated into a single unit; suitable materials may includesemi-flexible plastics such as polypropylene. The unit can be preparedby machining or moulding.

The use of rigid or semi-rigid materials in the component parts of thebag allows for the formation or compression of the packed bed.Preferably, the bag and its component parts are fabricated frombiologically and chemically inert materials which are approved byregulatory authorities, such as the US Food and Drug Administration, forthe manufacture of drugs.

The bag (10) may also comprise a second compartment (17), adjacent tothe first compartment (11) of the bag (10) which acts as a hydraulicchamber to compress the first compartment (11) as described below. Inother embodiments, the second compartment (17) may be a separate bagadjacent to the first compartment (11) which acts as a hydraulicchamber.

In FIGS. 1a and 1b , the bag (10) is enclosed by the column (20) housing(22) and lid (21), at least one of these components being semi-rigid orrigid. The bag (10) is filled with a dry powder or wet slurry ofchromatography media through port 16. The column (20) may be sterilisedby gamma irradiation on filling with media prior to use, oralternatively the bag may be sterilised by gamma irradiation orautoclaving on charging with media, and then stored or shipped to auser.

To prepare the media bed for chromatographic separation, a user willinsert the the bag (10) into a chamber of a chromatography column (20),close the column housing (22) and lid (21) such that walls of the bag(10) fit tightly within the chamber and, in one embodiment, use the flowpacking method to compress the bed (not shown). The packed column couldthen also be sterilised by gamma radiation prior to use.

In another embodiment, the second compartment or hydraulic chamber (17)can be filled with hydraulic fluid via port (24) to axially compress thefirst compartment (11) of the bag (10) containing media in order tomaintain bed stability.

Alternatively, the first compartment (11) can be compressed mechanicallyby a piston or adapter present in the column chamber.

Separation of chemical compounds, such as proteins, may now occur byflowing a carrier liquid containing the compounds into the bag 10 (viainlet 25) and through the chromatographic media, the eluant beingcollected at outlet 26. Alternatively, the carrier flow can be reversed,such that port 26 serves as an inlet and eluant is collected at port 25.

In another embodiment, the user will insert the bag (10) into a chamberof an axial chromatography column (20), close the column housing (22)and lid (21) such that the bag is confined by the rigid housing andarrange the column in a vertical direction to apply liquid to the bagsuch that a consolidated bed is formed. When forming the consolidatedbed by application of flow in downward direction, a gap may be formedabove the consolidated bed.

In another embodiment, the walls of the bag will be displaced to reducesaid gap formed above the consolidated bed in order to reduce thehold-up volume of column and/or to mechanically stabilise theconsolidated bed.

In use, a liquid sample containing a chemical (such as a protein,peptide, antibody, nucleic acid or mixtures thereof) or a cell (orcellular mixture) is loaded onto the consolidated bed and allowed toequilibrate. The bag is then removed from the column and may be storedat low temperature (e.g. −20.degree. C.) to minimise chemical orcellular loss or breakdown due to chemical, photochemical or biologicalinstability. Alternatively, the bag may be transported at lowtemperature (such as, for example, −20.degree. C.) to another location.Following storage and/or transport at low temperature, the bag may beequilibrated to an operating temperature at or above 4.degree. C.,placed in a semi rigid or rigid column and the chemical or cell elutedfrom the chromatographic medium. Alternatively, the chemical or cellwhich is loaded on the chromatographic medium may be subjected toprocessing, such as a chemical or biological reaction, followingequilibration and replacement of the bag in the rigid or semi-rigidcolumn at temperatures at or above 4.degree. C. The product of theprocessing step may then be eluted from the column.

In one embodiment, the axial column of FIG. 1 is designed in acylindrical shape. In another embodiment, the axial column is designedhaving a cross-sectional shape deviating from a circle found at thecylindrical shape, such as being of oval or rectangular shape. The shapeof the rigid housing confining the column is adapted accordingly. Forexample, the column could be made to adopt to the shape of a 2 D pillowbag or a 3D rectangular bag that has been fitted with suitabledistribution systems at inlet and outlet side. In yet anotherembodiment, the cross section of the column and bag may vary across theaxial position in between the end pieces as being rectangular or oval atthe end pieces and being circular or oval in the middle of the column,for example.

In another embodiment, the rigid housing is opened and closed at theside of the column and housing instead of having a removable lid asdepicted in FIG. 1. In yet another embodiment, the rigid housingconsists of two or more wall segments mounted together after havinginserted the bag.

FIGS. 2a, b and c are schematic sectional plan views of a bag (210) inaccordance to the invention which can be packed by radial compression.The bag (210) is tubular in nature with a hollow core or centre (230).In the embodiment shown, the exterior (212) and interior (219) walls ofthe bag (210) have welded opposed liquid distribution elements (213,214) which allow passage of carrier liquid from the exterior to theinterior of the bag (or in the reverse direction depending upon thedirection of flow). In the embodiment shown, the bag (210) hassemi-rigid walls (222) which can exert a radial, compressive forceagainst the walls (212, 219) of the bag (210). The outer or exteriorwall (212) of the bag has indents (215) which allow mild compression ofthe media filled bag (210) in response to the radial force of thesemi-rigid walls (222). The bag (210) is stable under such mildcompression and may be stored or transported to a user (FIG. 2a ).

Bed packing is achieved by applying a radial mechanical compression tothe walls (222) of the bag (210) as indicated by the arrows in FIG. 2b .As can be seen in FIG. 2c , the radial forces exerted on the bag (210)closes the indents (215) and is transmitted through the bag, compressingthe media within.

In another embodiment, mild compression of bed is achieved using theflow packing method described above.

FIGS. 3a, b and c show the component parts of a radial column using abag 310 according to the invention. FIG. 3a illustrates a cylindricalrigid core (340) with a stand (342) and locking lid (344). The lid 344has apertures or connectors to accommodate an outlet port (346) foreluant and a media filling port (348). FIG. 3b depicts an outercylindrical shell (350), typically composed of a semi rigid plasticsheet (351), which can wrapped around the radial flow bag 310 shown inFIG. 3c and used to radially compress it. In the embodiment shown, thelength of the sheet (351) is greater than the external circumference ofthe bag 310 such that there is an overlap 354 to allow for changes inthe diameter of the sheet (351) during compression. Other functionaldesigns, such as pleating of the sheet, are also possible to achievethis purpose. The shell (350) has an aperture or connector (352) toaccommodate a carrier liquid inlet (325).

FIG. 3c illustrates a bag (310) according to the invention. The bag hasa tubular configuration with a hollow core 330. A first liquiddistribution element (313) and second liquid distribution element (314)are welded to the exterior (312) and interior (319) walls of the bag(310), respectively. A media inlet port (316) is used to fill the bagwith media, typically in the form of a slurry, as indicated by the arrowin FIG. 3c . Carrier liquid enters the bag via inlet 325, flows throughthe media bed (not shown) and exits via outlet (326, see arrows).

A radial flow column (360) is prepared by inserting the bag (310) overthe rigid core (340), wrapping the cylindrical shell (350) around theexterior wall of the bag, and locking the lid (344) into position (FIG.3d ). The bag is filled with media slurry via inlet port 316 and excessmedia removed via outlet 326 or 325, prior to sealing the inlet port(316). The media in the bag may then be equilibrated with carrier liquidwhich will enter the column via inlet 325 and exit via port 326. In oneembodiment, the bed is compressed by the flow packing method describeabove.

In another embodiment, the outer shell (350) is radially compressed toform a packed bed of chromatography media in the bag. Radial compressioncan be effected in a number of ways, such as pulling the overlap 354 toconstrict and compress the bag against the rigid core (FIG. 3e ). Othermeans for compression are possible, such as clamping or tighteningradial bands. Once the outer shell 350 has been compressed it is securedin position to stabilise the packed bed of media (for example, by use oftie bands 370). The column is now ready to be used for chromatographicseparation of chemicals, such as proteins and/or peptides. The samplecontaining the chemical(s) to be separated or purified are dissolved incarrier liquid and applied to the column. The carrier liquid enters thebag via inlet 325 and first distribution element 313, partitions overthe media bed (not shown) where separation occurs and passes through thesecond element 314 to exit the column at outlet port 326. It will beunderstood, that the column may be run in the reverse direction byreversing the direction of flow.

Low Temperature Storage Experiments

The experiments detailed below are described fully in Applicant'sco-pending U.S. patent application 61/370,878 entitled “Stabilizationand Storage Media and Method”, filed on 5 Aug. 2010. Where national lawpermits, the content is hereby incorporated by reference in its entiretyherein as if it had been specifically incorporated.

It is generally known that the storage of proteins (e.g. antibodies) insolution leads to aggregation over time. By freezing the proteinsolutions in a carefully designed buffer environment at slow andcontrolled rates of freezing aggregation can be reduced, but theaggregate content will inevitably be higher after storage. Experimentswere then performed using an alternative storage approach by usingdifferent gel media to bind or encapsulate the target proteins.

Storage of Human Polyclonal IgG with a Low Initial Dimer Content inSolution or on Gel Media

The monomer fraction of human polyclonal IgG (1% dimer) was firstconcentrated to 121.5 AU (A280 nm) using VIVASPIN® 6 ultrafiltrationunits and filtered with 0.2.mu.m syringe filters. Various experimentalcapture storage hydrogel media, and control (noncapture SEC) media (seeMaterials above) were dispensed into 96 well filter plates (MULTITRAP®plates). The MULTITRAP® protocol was followed using the concentratedmonomer fraction of human polyclonal IgG as antibody sample. Inparallel, antibody samples were mixed with the various buffers used forthe different “gel media” for subsequent storage in solution at−20.degree. C. 6.7 .mu.l antibody solution was mixed with 13.3 .mu.l ofeach buffer respectively.

Buffers used for samples and equilibration/washing of gel media weretypical adsorption capture buffers for the different media used:

“pH 5 IEX”=20 mM Na-Acetate, 0.02% (w/v) Na-azide, pH 5.0

“pH 9 IEX”=20 mM Na-Glycine, 0.02% (w/v) Na-azide, pH 9.0

“PBS”=10 mM Na-Phosphate, 2.7 mM KCl, 0.14M NaCl, 0.02% (w/v) Na-azide,pH 7.4

“pH 5 HIC”=25 mM Na-Acetate, 0.5 mM EDTA, 0.75M (NH4)2SO4, pH 5.0

The various chromatography gel media were stored in the 96-wellMULTITRAP® plate. The MULTITRAP® plates were stored in the fridge for 4weeks at 4-8.degree. C. (the protocol incubation time), whereas theantibody samples in solution were stored at −20.degree. C. for 4 weeksincluding 6 freeze/thaw cycles. All samples were run in triplicateexcept for three of the anion exchange gel media, CAPTO® Q, Q SEPHAROSE®FF and Q SEPHORASE® XL, that were run as single samples. Afterincubation or storage for 4 weeks, either the non-bound antibodies(flow-through) were collected (gel filtration SEC media SEPHADEX® G-50and SUPERDEX® 200) or the bound and then eluted antibodies (all mediaexcept gel filtration media). Elution and strip buffers used were:

elution buffer: “3.3.times.PBS”=30 mM Na-Phosphate, 8.1 mM KCl, 0.42 MNaCl, pH 7.4 (for those samples of which buffers used for samples andequilibration/washing of gel media were “pH 5 IEX” and “pH 9 IEX”);

elution buffer: 0.1 M Na-Glycine pH 2.9 (for those samples of whichbuffers used for samples and equilibration/washing of gel media were“PBS);

elution and strip buffer: 20 mM Tris-HCl pH 7.5 (for those samples ofwhich buffers used for samples and equilibration/washing of gel mediawere “pH 5 HIC”);

strip buffer: 10 mM NaOH, 1M NaCl for those samples of which buffersused for samples and equilibration/washing of gel media were (“pH 5 IEX”and “pH 5 HIC”);

strip buffer: 0.5M Acetic acid (for those samples of which buffers usedfor samples and equilibration/washing of gel media were “PBS” and “pH 9IEX”)

Elution was performed in two steps, first with elution buffers andsecondly with strip buffers. The latter are typically in bioprocessingused to remove any residual protein not eluted by the elutionbuffers—and to prepare column for possible additional use. Both elutionfractions for each sample were analyzed respectively. All samples,stored in solution, on SEC control media or on binding media, wereanalyzed by SEC. The results are shown in Tables 1A-1D.

TABLE 1A Dimer content in eluted fractions from human polyclonal IgGstored on gel media at 4-8° C. or frozen in solution at −20° C. for 4weeks in pH 5 “IEX” buffer (20 mM Na-Acetate, 0.02% (w/v) Na-azide, pH5.0). % dimer replicate comment Gel media CAPTO ® MMC 1.72 1 CAPTO ® MMC0.84 2 CAPTO ® MMC 0.75 3 CAPTO ® S 2.95 1 CAPTO ® S 2.53 2 CAPTO ® S1.68 3 SP SEPHAROSE ® FF 2.68 1 SP SEPHAROSE ® FF 2.21 2 SP SEPHAROSE ®FF 2.58 3 SP XL 2.44 1 SP XL 1.84 2 SP XL 1.72 3 Storage in solution(−20° C.) pH 5 “IEX” 3.41 1 pH 5 “IEX” 3.23 2 pH 5 “IEX” 3.92 3

All samples stored on these gel media contained less antibody dimercontent compared with storing the samples in solution at −20.degree. C.The start material contained 1.0% dimer and two of the replicates usingCAPTO® MMC as storing gel media contained less dimer than this initialdimer content level.

TABLE 1B Dimer content in eluted fractions from human polyclonal IgGstored on gel media at 4-8° C. or frozen in solution at −20° C. for 4weeks in “PBS” buffer (10 mM Na-Phosphate, 2.7 mM KCl, 0.14M NaCl, 0.02%Na-azide, pH 7.4). % dimer replicate comment Gel media nProtein A 0.31 1SEPHAROSE ® 4 FF nProtein A 0.30 2 * <0.3, difficult to SEPHAROSE ® 4 FFintegrate peaks nProtein A 0.30 3 * <0.3, difficult to SEPHAROSE ® 4 FFintegrate peaks MABSELECT ® 0.52 1 MABSELECT ® 0.50 2 * <0.5, difficultto integrate peaks MABSELECT ® 0.50 3 * <0.5, difficult to integratepeaks SUPERDEX ® 200 4.25 1 SUPERDEX ® 200 4.54 2 SUPERDEX ® 200 4.14 3SEPHADEX ® G-50 4.93 1 SEPHADEX ® G-50 5.18 2 SEPHADEX ® G-50 4.94 3Storage in solution (−20° C.) PBS, pH 7.4 3.24 1 PBS, pH 7.4 3.27 2 PBS,pH 7.4 3.00 3

Samples stored on the affinity gel media contained less antibody dimercompared with storing the samples in solution at −20.degree. C. However,storage on noncapture SEC media resulted in higher levels of dimer. Thestart material contained 1.0% dimer and using nProtein A or MABSELECT®as storing gel media resulted in less dimer content than the initialdimer content.

TABLE 1C Dimer content in eluted fractions from human polyclonal IgGstored on gel media at 4-8° C. or frozen in solution at −20° C. for 4weeks in “pH 5 HIC” buffer (25 mM Na-Acetate, 0.5 mM EDTA, 0.75M(NH4)2SO4, pH 5.0). % dimer replicate comment Gel media ButylSEPHAROSE ® HP 1.31 1 Butyl SEPHAROSE ® HP 1.05 2 Butyl SEPHAROSE ® HP1.25 3 CAPTO ® Phenyl high sub. 0.10 1 * <0.1 CAPTO ® Phenyl high sub.0.10 2 * <0.1 CAPTO ® Phenyl high sub. 0.00 3 pH HIC 2% poor binding ofsample at 0.75M AmSO pH HIC 2% poor binding of sample at 0.75M AmSO pHHIC 2% poor binding of sample at 0.75M AmSO pH HIC 4% AMBN poor bindingof sample at 0.75M AmSO pH HIC 4% AMBN poor binding of sample at 0.75MAmSO pH HIC 4% AMBN poor binding of sample at 0.75M AmSO SEPHAROSE ® HP16% poor binding of sample AMBN at 0.75M AmSO SEPHAROSE ® HP 16% poorbinding of sample AMBN at 0.75M AmSO SEPHAROSE ® HP 16% poor binding ofsample AMBN at 0.75M AmSO CAPTO ® Phenyl low sub. 1.15 1 CAPTO ® Phenyllow sub. 0.87 2 CAPTO ® Phenyl low sub. 1.26 3 SEPHAROSE ® HP 16% poorbinding of sample AMBN at 0.75M AmSO SEPHAROSE ® HP 16% poor binding ofsample AMBN at 0.75M AmSO SEPHAROSE ® HP 16% poor binding of sample AMBNat 0.75M AmSO Storage in solution (−20° C.) pH 5 “HIC” 1.55 1 pH 5 “HIC”1.74 2 pH 5 “HIC” 1.69 3

The overall recoveries from the HIC gel media were poor, probably due topoor binding of the antibodies due to using too low a conductivity (saltconcentration) in the adsorption buffer. To promote binding to the HICgel media a higher concentration of (NH4) 2SO4 was needed. The resultsthat were obtained showed that binding to the gel media gives less dimerduring storage compared with storage in solution and for CAPTO® Phenylhigh substitution (higher ligand concentration) media the dimer contentwas less than 1.0% (level in the start material).

TABLE 1D Dimer content in eluted fractions from human polyclonal IgGstored on gel media at 4-8° C. or frozen in solution at −20° C. for 4weeks in “pH 9 IEX” buffer (20 mM Na-Glycine, 0.02% (w/v) Na-azide, pH9.0). % dimer replicate comment Gel media CAPTO ® Adhere 0.69 1 CAPTO ®Adhere 0.95 2 CAPTO ® Adhere 0.75 3 CAPTO ® Q 2.27 1 CAPTO ® Q no sampleCAPTO ® Q no sample Q SEPHAROSE ® FF 3.84 1 Q SEPHAROSE ® FF no sample QSEPHAROSE ® FF no sample Q XL 3.31 1 Q XL no sample Q XL no sampleStorage in solution (−20° C.) pH 9 “IEX 3.39 1 pH 9 “IEX 3.35 2 pH 9“IEX 3.58 3

Samples stored on CAPTO® Adhere gel media contained less antibody dimercontent compared with storing the samples in solution at −20.degree. C.The dimer content was less than 1.0% as found in the start material.Storage on the other gel media resulted in about the same levels ofdimeric proteins as storage in solution.

Storage of human polyclonal IgG with a high initial dimer content insolution or on gel media

Polyclonal human IgG (GAMMANORM® 165 mg/ml) was diluted to 30 mg/ml withfollowing equilibration/wash buffers:

20 mM Na-Acetate, 0.02% (w/v) Na-azide, pH 5.0

20 mM Na-Glycine, 0.02% (w/v) Na-azide, pH 9.0

10 mM Na-Phosphate, 2.7 mM KCl, 0.14M NaCl, 0.02% (w/v) Na-azide, pH 7.4

50 mM Na-Acetate, 1 mM EDTA, 1.5M (NH4) 2SO4, pH 5.0

Some protein was precipitated upon mixing the antibody solution with thefourth buffer solution containing 1.5M (NH4) 2504. The absorbance ofthese start materials were measured (on clarified solutions):

Sample buffer A 280 cm⁻¹ 20 mM Na Acetate, 0.02% (w/v) Na azide, pH 5.040.6 20 mM Na Glycine, 0.02% (w/v) Na azide, pH 9.0 40.5 10 mM NaPhosphate, 2.7 mM KCl, 0.14M NaCl, 0.02% 40.8 (w/v) Na azide, pH 7.4 50mM Na Acetate, 1 mM EDTA, 1.5M (NH₄)₂SO₄, pH 28.5 5.0

SPINTRAP® columns were filled with 40 .mu.l of following gel media (i.e.200 .mu.l 20% gel slurry) and were equilibrated according to theSPINTRAP® protocol:

CAPTO® MMC mixed mode media, 20 mM Na-Acetate, 0.02% (w/v) Na-azide, pH5.0

CAPTO® S cation exchange media, 20 mM Na-Acetate, 0.02% (w/v) Na-azide,pH 5.0

CAPTO® Adhere mixed mode media, 20 mM Na-Glycine, 0.02% (w/v) Na-azide,pH 9.0

CAPTO® Q anion exchange media, 20 mM Na-Glycine, 0.02% (w/v) Na-azide,pH 9.0

MABSELECT®, affinity media, 10 mM Na-Phosphate, 2.7 mM KCl, 0.14M NaCl,0.02% (w/v) Na-azide, pH 7.4

SEPHADEX® G-50, control SEC media, 10 mM Na-Phosphate, 2.7 mM KCl, 0.14MNaCk, 0.02% (w/v) Na-azide, pH 7.4

CAPTO® Phe hs, phenyl ligand containing HIC media, 50 mM Na-Acetate, 1mM EDTA, 1.5M (NH.sub.4).sub.2SO.sub.4, pH 5.0

pH HIC 6%, pH responsive polymer based HIC media, 50 mM Na-Acetate, 1 mMEDTA, 1.5M (NH.sub.4).sub.2SO.sub.4, pH 5.0

pH HIC 16%, pH responsive polymer based HIC media, 50 mM Na-Acetate, 1mM EDTA, 1.5M (NH.sub.4).sub.2SO.sub.4, pH 5.0

After equilibration, 40 .mu.l of sample was added to each column(matching buffers). Three SPINTRAP® columns of each gel media containingbound antibodies were stored at room temperature (+20.degree. C.), infridge (+4-8.degree. C.) and in the freezer (−20.degree. C.)respectively. In parallel, aliquots of antibodies in solution containingthe various buffers were also stored at the same temperatures as theSPINTRAP® columns.

60 .mu.l of matching buffer was added to all SPINTRAP® columns after anincubation time of 24-26 days. The flow-through (non-binding fraction)was collected and the absorbance (A280 nm) was measured.

The various gel media was washed, then eluted with 400 .mu.l offollowing elution buffers:

CAPTO® MMC, 20 mM Na-Phosphate, 1M NaCl, pH 7.0

CAPTO® S, 20 mM Na-Phosphate, 1M NaCl, pH 7.0

CAPTO® Adhere, 0.5M Acetic acid [0118] CAPTO® Q, 20 mM Na-Phosphate, 1MNaCl, pH 7.0

MABSELECT®, 0.5M Acetic acid

SEPHADEX® G-50, not eluted, flow-through fraction collected

CAPTO® Phe hs, 20 mM Na-Phosphate, 1M NaCl, pH 7.0

pH HIC 6%, 20 mM Na-Phosphate, 1M NaCl, pH 7.0

pH HIC 16%, 20 mM Na-Phosphate, 1M NaCl, pH 7.0

The absorbance (A280 nm) was measured on the eluted fractions. Allsamples from storage in solution and flow-through/elution fractions fromthe various gel media were analyzed by SEC. The antibody recovery andthe dimer content are summarized in Table's 2A-4D.

TABLE 2A Antibody recovery and dimer content in eluted fractions afterstorage of human polyclonal IgG on gel media or in solution. MonomerTemp. % dimer after Total protein IgG ° C. 3.5 weeks recovery % recovery% Storage on gel media CAPTO ® 20 8.0 24 27 MMC 20 mM 5 11.1 71 82acetate pH 5.0 −20 10.7 76 87 CAPTO ® 20 6.4 87 98 S 20 mM 5 8.5 93 108acetate pH 5.0 −20 8.3 88 101 In solution storage 20 mM 20 11.6 acetatepH 5.0 5 13.8 −20 13.0 Buffer for storage: 20 mM Na-Acetate, 0.02% (w/v)Na-azide, pH 5.0. Initial dimer content directly after dilution was16.9%.

Storage on CAPTO®S results in much reduced dimer content and theantibody recovery is high. CAPTO® MMC gives slightly lower dimer contentafter storage compared with storage in solution with good recoveries atlower temperatures.

TABLE 2B Antibody recovery and dimer content in eluted fractions afterstorage of human polyclonal IgG on gel media or in solution. MonomerTemp. % dimer after Total protein IgG ° C. 3.5 weeks recovery % recovery% Storage on gel media CAPTO ® 20 4.0 88 111 Adhere 20 mM 5 1.5 93 122glycine pH 9 −20 2.0 93 109 CAPTO ® 20 7.4 72 91 Q 20 mM 5 9.0 57 74glycine pH 9 −20 7.5 66 77 In solution storage 20 mM 20 20.9 glycine pH9 5 23.5 −20 14.5 Buffer for storage: 20 mM Na-Glycine, 0.02% (w/v)Na-azide, pH 9.0. Initial dimer content directly after dilution was24.4%.

Storage on CAPTO® Adhere gives much improved results compared withstorage in solution. The recoveries are very high with possibleconversion of dimers into monomers as evidenced by the higher than 100%recovery of monomer IgG. Results from storage on CAPTO® Q show lessdimer content compared with storage in solution, but the recoveries wereless than 80%.

TABLE 2C Human polyclonal IgG antibody recovery and dimer content ineluted fractions after storage. Monomer Temp. % dimer after Totalprotein IgG ° C. 3.5 weeks recovery % recovery % Storage on gel mediaMABSELE 20 1.7 88 108 CT ® PBS, pH 5 2.2 89 115 7.4 −20 2.1 90 116SEPHADE 20 14.4 98 120 X ® G-50 PBS, pH 5 17.0 73 94 7.4 −20 14.4 97 125In solution storage PBS, pH 20 18.3 7.4 5 22.4 −20 22.3 Buffer forstorage: 10 mM Na-Phosphate, 2.7 mM KCl, 0.14M NaCl, 0.02% (w/v)Na-azide, pH 7.4. Initial dimer content directly after dilution was24.6%.

Storage on MABSELECT® gives much improved results compared with storagein solution. The recoveries are very high with possible conversion ofdimers into monomers evidenced by the higher than 100% recovery ofmonomer IgG.

Results from storage on SEPHADEX® G-50 show slightly less dimer contentcompared with storage in solution but have the highest protein recoveryof all gel media perhaps due to the non-binding properties.

TABLE 2D Human polyclonal IgG antibody recovery and dimer content ineluted fractions after storage. Monomer Storage on Temp. % dimer afterTotal protein IgG gel media ° C. 3.5 weeks recovery % recovery % CAPTO ®20 6.5 31 Phe hs 5 5.7 49 −20 7.7 57 pH HIC 20 5.9 70 16% 5 6.9 79 −209.0 68 pH HIC 20 5.7 68 6% 5 7.4 80 −20 8.2 82 Buffer for storage: 50 mMNa-Acetate, 1 mM EDTA, 1.5M (NH₄)₂SO₄, pH 5.0. Initial dimer contentdirectly after dilution was not measured due to precipitation.

For HIC gel media, there was no starting material for comparison due totarget precipitation. The absorbance (A280 nm) of the start material was28.5 AU compared with over 40 AU for the other start materials. Theprotein recovery after clarification was good for pH HIC 6% and pH HIC16% gel media. Compared with other gel media, the dimer content afterstorage was in the low range, showing a positive effect on aggregationstabilization or reduction.

The results for all storage conditions are shown as graphs in FIGS.4A-C. Storage on CAPTO®Adhere or on MABSELECT® shows very low levels ofdimers or aggregates with high recoveries of monomer IgG after storage,reflecting apparent ability of the media to reduce dimer levels duringstorage. Storage on CAPTO® Adhere or on MABSELECT® shows very low levelsof dimers or aggregates with high recoveries of monomer IgG afterstorage, reflecting apparent ability of the media to reduce dimer levelsduring storage.

The initial dimer content directly after dilution of GAMMANORM® (165mg/ml) was between 16.9-24.6% depending on buffer used. After storagefor 3.5 weeks at different temperatures, the dimer content was slightlylower in the various solutions.

The buffer composition, pH, salt content and type seem to affect theequilibrium faster and to a higher degree than compared with the proteinconcentration. Storage of GAMMANORM® in a low salt buffer at pH 5.0results in much lower dimer content compared with storage at pH 7.4 orpH 9.0, both directly after dilution and after storage for 3.5 weeks.

The effect of temperature is less obvious.

The best storage condition for GAMMANORM® in solution seems to bestorage in 20 mM Na-Acetate, 0.02% Na-azide pH 5.0 at +20.degree. C.according to these experiments.

However, the most pronounced effect on dimer content was observed whenstoring antibodies on binding media. Especially on CAPTO™ Adhere andMABSELECT1.upsilon., although storage on these gel media was done at pH9.0 and pH 7.4 respectively. The effect was so great, that whencalculating the recovery of monomeric IgG, the result was higher than100% for samples stored on CAPTO®Adhere and MABSELECT® The total proteinrecovery was never higher than 98% though (SEPHADEX® G-50, non-bindingmedia).

Thus, storing on binding media may have several benefits including:

1. Stabilization of IgG against aggregation (slow down kinetics)

2. Removal of IgG dimer (polishing)

3. Promote monomer formation (reversal of monomer/dimer equilibrium)

Storing a start material with low initial dimer content on gel mediawould benefit from stabilization and polishing. Starting from highinitial dimer content would also benefit from reducing the amount ofdimers and increase the percent of monomers.

Results obtained from non-binding media show no or little reduction ofdimer content. In the first experiment starting with low initial dimercontent, the storage on non-binding gel media (SUPERDEX®200 andSEPHADEX®G-50) resulted in slightly more dimers compared with storing insolution. Starting with high initial dimer content, the storage onSEPHADEX®G-50 resulted in slightly less dimers compared with storage insolution. The overall dimer content was however always higher comparedwith storing on any capture gel media.

The skilled person will understand that flexible bags in accordance tothe invention may be employed for the preparation of separation orreaction units that rely on introducing a particulate material into acolumn chamber and bringing the material into contact with a fluid aseither a packed, expanded, consolidated or fluidized bed for achievingthe separation or reaction process.

Whilst the present invention has been described in accordance withvarious aspects and preferred embodiments, it is to be understood thatthe scope of the invention is not considered to be limited solelythereto and that it is the Applicant's intention that all variants andequivalents thereof also fall within the scope of the appended claims.

The invention claimed is:
 1. A flexible bag for chromatographic mediumfor insertion into a chromatographic column housing, said flexible bagcomprising: an exterior wall of a non-porous material and a separateinterior wall which is concentric to the exterior wall and spaced apartto define a compartment for chromatographic medium therein; the exteriorwall is attached to both a first liquid distribution element and asecond liquid distribution element at respective opposed ends of saidexterior wall; said first liquid distribution element facing said secondliquid distribution element, the first liquid distribution element beingwelded or moulded to the exterior wall; a medium filling port; and afirst end piece and a second end piece attached to said exterior walland adjacent to the first liquid distribution element and the secondliquid distribution element respectively, the first end piece and thesecond end piece each comprising an opening for receipt of an inlet oran outlet for carrier liquid and wherein the first liquid distributionelement and the second liquid distribution element is each selected fromthe group consisting of filter, mesh, net, and sinter, and each of thefirst and second liquid distribution elements includes pores that areporous to liquids and do not allow the chromatographic medium to passthrough.
 2. The flexible bag for chromatographic medium of claim 1,wherein said flexible bag has a tubular configuration.
 3. The flexiblebag for chromatographic medium of claim 1, wherein the first liquiddistribution element and the first end piece are an integrated unit, andthe second liquid distribution element and the second end piece are anintegrated unit.
 4. The flexible bag for chromatographic medium of claim1, wherein said flexible bag further comprises a second compartmenthaving an inlet for hydraulic fluid.
 5. A flexible bag forchromatographic medium for insertion into a chromatographic columnhousing, said flexible bag comprising: an exterior wall of a non-porousmaterial and a separate interior wall which is concentric to theexterior wall and spaced apart to define a compartment forchromatographic medium therein, and the flexible bag being formed of atubular configuration; a first liquid distribution element facing asecond liquid distribution element, the first liquid distributionelement being welded or moulded to the exterior wall and the secondliquid distribution element being welded or moulded to the interiorwall; wherein the first liquid distribution element and the secondliquid distribution element each includes pores that are porous toliquids and do not allow the chromatographic medium to pass through achromatographic medium filling port; and a first end piece and a secondend piece attached to said exterior wall and adjacent to the firstliquid distribution element and the second liquid distribution elementrespectively, the first end piece and the second end piece eachcomprising an opening for receipt of an inlet or an outlet for carrierliquid.